Immunoreactivity of glutamine synthetase in satellite glia around various subpopulations of lumbar dorsal root ganglia neurons in adult rats treated with monosodium glutamate.

Satellite glial cells (SGCs), involved inter alia in glutamate (Glu) metabolism, form a glial sheath around sensory neurons of dorsal root ganglia (DRGs). SGCs show a presence of glutamine synthetase (GS) which transform uptaken Glu into glutamine (Gln). In DRGs, this aminoacid is used mainly by small neurons which are able to synthetize substance P (SP) that play a crucial role in nociception. The aim of the study was to define the influence of monosodium glutamate (MSG) on GS immunoreactivity in satellite glia around various subpopulations of neurons including SP immunopositive cells in DRGs of adult rats. The studies were carried out on lumbar DRGs slides in rats which received subcutaneous injection of saline solution (control group) or 4 g/kg b. w. of MSG (MSG group). Immunofluorescence reactions were conducted with use of anti-GS and anti-SP antibodies. Administration of MSG to adult rats increased the GS immunoexpression in SGCs. In rats receiving MSG, a number of small neurons with GS-immunopositive glial sheath was not altered when compared to control individuals, whereas there was a statistically significant increase of GS immunoexpression in SGCs around large and medium neurons. Moreover, in these animals, a statistically significant increase in the number of small SP-positive neurons with GS-positive glial sheath was observed. SP is responsible for transmission of pain, thus the obtained results may be useful for further research concerning the roles of glia in nociceptive pathway regulation.

Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

Insulin receptors in the CA1 field of hippocampus and selected blood parameters in diabetic rats fed with bilberry fruit.

INTRODUCTION: Bilberry fruit is believed to be a promising factor in the treatment of diabetes mellitus. Chronic hyperglycaemia affects the function of the central nervous system, which may be manifested as changes in hypothalamic insulin signalling. MATERIAL AND METHODS: Using DPPH and ABTS assays, total phenolic content in bilberry fruit and its antioxidant activities were examined. The selected biochemical parameters of blood (glucose, fructosamine, total cholesterol, HDL-cholesterol, LDL-cholesterol and triglycerides), as well as the expression of insulin receptors, were studied in the hippocampal CA1 field of healthy and diabetic (streptozotocin-induced; 60 mg kg-1 body weight) Wistar rats fed with bilberry fruit (16 g kg-1 body weight per day; 6 weeks), as well as of the corresponding control groups. RESULTS: Biochemical analyses revealed ambiguous results, but a significantly (P<0.05) decrease in the level of LDL-cholesterol was observed in the group of healthy rats supplemented with bilberry pulp after 6 weeks of the treatment. There was also a difference (P<0.05) in the level of LDL-cholesterol in the mentioned healthy animals fed with bilberry, versus the healthy control group. An increased number of insulin receptors-immunoreactive neurons as well as nerve fibres in the CA1 field of diabetic rats fed with bilberry fruit was also found. CONCLUSIONS: An inclusion of bilberry fruit in the daily diet during the course of diabetes can lead to plasticity of hippocampal neurons/nerve fibres, manifested by changes in insulin receptors expression. Whether or not the observed changes had protective effects (by reducing damages caused by diabetes mellitus) on the function of the central nervous system neurons needs further study.

Modern Hybrid Rye, as an Alternative Energy Source for Broiler Chickens, Improves the Absorption Surface of the Small Intestine Depending on the Intestinal Part and Xylanase Supplementation.

The current study investigated the effects of the inclusion of modern hybrid rye (Brasetto variety) to a corn-wheat-based diet, with or without xylanase, on the absorptive surface of the small intestine of broilers. A total of 224 one-day-old male Ross 308 broiler chicks were randomly divided into four experimental groups with seven replicate cages of eight birds/replicate. A 2 × 2 factorial study design was used, with rye inclusion (0% or 20%) and xylanase supplementation (0 or 200 mg/kg of feed) as factors. Inclusion of rye increased duodenal and ileal crypt depth, villi height, the villus-to-crypt ratio and absorption surface area (p < 0.05), and ileal mucosa thickness and crypt width (p < 0.05). Xylanase supplementation attenuated the effects of rye in the duodenum and ileum and decreased the villi height and villus-to-crypt ratio in the jejunum (p < 0.05). Rye and xylanase had no effect on the spatial distribution of claudin 3 and ZO-1 protein, but xylanase supplementation reduced the amount of claudin 3 in the duodenum and jejunum (p < 0.05). The findings of this study indicate that 20% inclusion of modern hybrid rye to the diets of broilers improved the structure of the duodenum and ileum, but these effects were attenuated by xylanase supplementation.

Co-expression patterns of cocaine- and amphetamine-regulated transcript (CART) with neuropeptides in dorsal root ganglia of the pig.

In the present study the neuronal distribution of CART was evaluated immunohistochemically in porcine dorsal root ganglia (DRGs). In co-localization studies the co-expression patterns of CART with SP, CGRP, galanin, CALB and LENK were investigated by means of triple immunohistochemical stainings. In porcine DRGs, the expression of CART was found in approximately 5% of primary sensory neurons. The vast majority (ca. 95%) of CART-immunoreactive (IR) neurons were small and middle sized, and only 5% were categorized as large. CART-IR neurons additionally exhibiting the presence of SP/CGRP (ca. 12%), SP/CALB (ca. 12%), SP/LENK (ca. 5%) were found. The vast majority of CART-IR/CGRP-IR neurons did not display immunoreaction to SP (ca. 60%). Subclasses of CART-IR/LENK-IR/SP-negative (ca. 5%), as well as CART-IR/CALB-IR/SP-negative neurons (ca. 10%), were also visualized. In addition, CART-IR neurons with no immunoreactivities to any of the neuropeptides studied were also shown. In porcine DRGs none of the CART-IR neurons exhibited the presence of galanin. The results obtained in the study suggest that CART may functionally modulate the activity of the porcine primary sensory neurons. It is concluded that co-expression of CART with CGRP, SP, LENK and CALB in subsets of the pig L1-L6 DRGs neurons provide anatomical evidence for a CART role in pain processing.

Immunolocalization of NOS, VIP, galanin and SP in the small intestine of suckling pigs treated with red kidney bean (Phaseolus vulgaris) lectin.

Lectins belong to a family of glycoproteins that can act both beneficially and detrimentally on the morphology of the small intestine. The aim of the study was to determine whether experimental treatment with red kidney bean (Phaseolus vulgaris) lectin influences the chemical code of the small intestine nervous system of suckling pigs. The immunolocalization sites of vasoactive intestinal polypeptide (VIP), nitric oxide synthase (NOS), substance P (SP) and galanin were determined in control and lectin-treated animals. In all segments of the small intestine (duodenum, jejunum, ileum), the subpopulations of VIP-, NOS-, SP- and galanin-immunoreactive (IR) myenteric neurons were unchanged. After lectin stimulation, increased proportions of NOS-IR and decreased numbers of VIP-IR submucous neurons/mucosa innervating nerve fibers were observed in the duodenum, jejunum and ileum. In lectin-treated animals down-regulation of submucous neurons expressing SP and up-regulation of galanin-IR submucous neurons were seen in the duodenum and jejunum (but not in the ileum). The distribution patterns of NOS-IR, galanin-IR and SP-IR nerve fibers supplying the duodenum, jejunum and ileum of the lectin-treated animals showed no substantial differences in relation to control piglets. We conclude that exposure to red kidney bean (P. vulgaris) lectin substantially changes the chemical content of VIP, NOS, SP and galanin in submucous neurons of the small intestine. These results are in line with previous findings outlining the key role(s) of these substances in enteric neuroplasticity processes and may constitute the basis for further functional studies on maturation of the gut.

Aquaporin 1 water channel is expressed on submucosal but not myenteric neurons from the ovine duodenum.

Aquaporins are a large family of small integral membrane proteins that function as molecular water channels. Increasing evidence indicates that an aquaporin 1 (AQP1) water channel is present on the surface of discrete neuronal classes of the central as well peripheral nervous systems. The aim of the present study has been to immunohistochemically localize AQP1 in the enteric nervous system (ENS) of the sheep duodenum. Specific antibodies to vasoactive intestinal polypeptide (VIP) and substance P (SP) were also used to biochemically determine possible function(s) of AQP1-positive enteric neurons. The expression of AQP1 in neuronal cell cultures has been also studied. Under normal conditions, approximately 30% of submucosal neurons exhibit the presence of AQP1 water channels. Neither myenteric neurons nor enteric nerve fibres showed immunoreactivity to AQP1. The vast majority of AQP1-bearing submucosal neurons were immunoreactive (IR) to SP (but not to VIP). Moderate numbers of SP-IR as well as VIP-IR nerve fibres run in close vicinity to AQP1-positive small blood/lymphatic vessels. SP-positive as well as VIP-positive nerve fibres were regularly observed to be in close contact with AQP1-positive submucosal neurons. After 3, 6 and 9 days of in vitro culturing, respectively, myenteric neurons still exhibited no presence of AQP1 channels. The obtained results indicated that in ENS of the ovine duodenum the expression of AQP1 is species-related and predominantly seen in a significant subpopulation of probably sensory submucosal neurons. Since we show no upregulation of AQP1 channels in cultured myenteric neurons we suggest that AQP1 is not a significant factor involved in environmental adaptation of myenteric neurons to the artificial conditions.

Distribution of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) nerve structures in the porcine large intestine.

The aim of the present study was to investigate the number of cocaine- and amphetamine-regulated transcript-like immunoreactive (CART-LI) nerve structures in the large intestine of juvenile pigs. The distribution pattern of CART-LI structures was studied by immunohistochemistry in the circular muscle layer, myenteric (MP), outer submucous (OSP) and inner submucous plexus (ISP) as well as in the mucosal layer of six regions of the large bowel: caecum, centripetal and centrifugal turns of the proximal colon, transverse colon, descending colon and rectum. CART-LI neural structures were observed in all gut fragments studied. CART-LI nerve fibres were numerous within the circular muscle layer and in the MP of all the regions studied, while they were moderate or few in number in other layers of the intestinal wall. The numbers of CART-LI neurons within the MP amounted to 2.02% in the caecum to 7.92% in the rectum, within the OSP from 2.73% in the centrifugal turns of the proximal colon to 5.70% in the rectum, and within the ISP from 2.23% in the transverse colon to 5.32% in the centrifugal turns of the proximal colon. The present study reports for the first time a detailed description of the CART distribution pattern within the enteric nervous system (ENS) of the porcine large intestine.

Immunohistochemical localization of galanin receptors (GAL-R1, GAL-R2, and GAL-R3) on myenteric neurons from the sheep and dog stomach.

Galanin exerts its biological activities (inhibitory or excitatory) via three different G protein-coupled receptors. In the present study, double immunocytochemical labeling was used to localize GAL-R1, GAL-R2 and GAL-R3 on PGP 9.5-positive myenteric neurons from the dog and sheep stomach/forestomachs. In both species, the occurrence of galanin in neurons and nerve fibers of gastric ganglia was also studied. Myenteric ganglia of the dog stomach were supplied with numerous, mainly varicose, galanin-immunoreactive (IR) nerve terminals whereas the frequency of galanin-positive nerve fibers in myenteric ganglia of the ovine stomach and forestomachs was moderate. The number of PGP 9.5-IR/galanin-IR myenteric neurons was significantly lower in the dog stomach (12.3+/-1.3%) as compared to the sheep rumen (20.1+/-0.7%), omasum (19.5+/-2.9%), abomasum (23.8+/-1.2%) but not reticulum (8.1+/-0.8%). In the canine stomach the frequencies of GAL-R1, GAL-R2 and GAL-R3 expressing myenteric neurons were statistically equivalent (4.4+/-0.9%, 3.5+/-0.7% and 3.1+/-0.5%, respectively). Immunoreactivity to GAL-R1 was absent in myenteric ganglia from the ovine rumen, reticulum as well as omasum. GAL-R1 was localized on 0.5+/-0.3% of myenteric perikarya from the abomasum. GAL-R2 bearing myenteric neurons were localized in the ovine rumen (0.6+/-0.3%), reticulum (0.5+/-0.3%), omasum (1.0+/-0.2%) and abomasum (1.1+/-0.3%). The percentages of PGP 9.5-IR/GAL-R3-IR neurons were 0.8+/-0.2% in the rumen, 0.6+/-0.3% in the reticulum, 0.7+/-0.2% in the omasum and 0.9+/-0.3% in the abomasum. In all compartments of the sheep stomach, the proportions of GAL-R1, GAL-R2 and GAL-R3 expressing neurons were significantly lower when compared to analogous neuronal subpopulations present in the dog. It is suggested that, although endogenous galanin may potentially inhibit or stimulate the activity of sparse gastric enteric neurons, its general role in indirect mediation of gastric motility and/or secretion seems to be of minor importance.

Cocaine- and amphetamine-regulated transcript (CART) is expressed in the ovine pancreas.

Indirect immunohistochemistry was applied to demonstrate the presence of cocaine- and amphetamine-regulated transcript (CART) peptide expression in the pancreas of the sheep. Using double immunocytochemical staining, the co-incidence of vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) or substance P (SP) in CART-immunoreactive (IR) nerve fibers and intrapancreatic neurons was analyzed. Immunoreactivity to CART was detected in endocrine cells predominantly localized at the islet periphery. The exocrine pancreas and blood vessels were intensively innervated by CART-IR nerve fibers. Moderate numbers of CART-IR nerve terminals were found in the connective tissue, while the ductal system and islets were poorly supplied with CART-IR nerve endings. No islet penetrating CART-IR nerve fibers were detected. Approximately 53.7+/-1.8% of intrapancreatic neurons displayed immunoreactivity to CART and pancreatic ganglia were moderately supplied with CART-IR nerve fibers. Dependent upon the pancreas region, CART-IR nerve fibers showed a varying degree of co-existence of SP, VIP or NPY. CART-IR intrapancreatic neurons very frequently co-localized with SP, moderately with VIP and rarely with NPY. We conclude that abundant immunoreactivity to CART in the ovine pancreas and the co-existence of CART with other regulatory peptides may reflect a possible involvement of CART in hormone and enzyme secretion as well as regulation of pancreatic blood flow.

A co-localization study on the ovine pancreas innervation.

The expression of DbetaH and several neuropeptides was investigated in neuronal elements of the ovine pancreas using double immunocytochemical stainings. Immunoreactivities to DbetaH, NPY, VIP and SP were seen to various extents in nerve terminals associated with the acini, islets, ducts, blood vessels, interlobular connective tissue as well as in the neurons of intrapancreatic ganglia. The expression of CGRP was limited to nerve fibers lying in the connective tissue septa, amongst the acini and in close vicinity to the pancreatic blood vessels. Single GRP-positive nerve endings were located around the acini, ducts and in the interlobular connective tissue. With the exception of the ductal system in a co-localization of NPY with DbetaH was frequently found in all regions of the pancreas. Moderately numerous blood vessel-associated VIP-positive nerve fibers as well as the vast majority of VIP-containing intrapancreatic neurons were found to co-express DbetaH. Single SP-immunoreactive (IR) nerve fibers of the exocrine pancreas and interlobular connective tissue as well as SP-positive intrapancreatic neurons additionally showed the presence of DbetaH. The co-localization of VIP and NPY was found in nerve terminals located around the blood vessels and acini, in the connective tissue septa as well as in numerous pancreatic neuronal perikarya. Rare nerve terminals located between the acini and around small blood vessels as well as several neurons of intrapancreatic ganglia were VIP-IR/ SP-IR. Simultaneous expression of SP and CGRP was found in nerve fibers supplying large pancreatic arteries and veins, interlobular connective tissue and, occasionally, around the acini. Throughout the pancreas the population of CGRP-positive nerve endings showed lack of VIP and NPY. In a moderate number of GRP-containing nerve fibers, a co-expression of NPY was noted. Nerve terminals containing both GRP and VIP were detected sporadically, whereas none of the GRP-positive nerve terminals showed expression of SP. We conclude that the presented noradrenergic as well as peptidergic innervation patterns of the ovine pancreas are species-dependent. On the basis of the occurrence of DbetaH, NPY, VIP and SP (alone or in combination) in pancreatic neuronal elements we can suggest that these substances presumably act as regulators of the endocrine and/or exocrine pancreas. Involvement of CGRP and GRP in the ovine pancreas physiology seems to be of minor importance. The co-localization study indicated that the pancreas of the sheep is innervated from several sources including intrinsic as well as extrinsic ganglia.

Neurochemical properties of the middle cervical ganglion in the sheep.

The neurochemical properties of the ovine middle cervical ganglion (MCG) were studied using antibodies raised against tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), neuropeptide Y (NPY), substance P (SP), calcitonin gene-related peptide (CGRP), vasoactive intestinal polypeptide (VIP) and galanin (GAL). Double-labelling immunocytochemistry revealed that the vast majority (95.5 +/- 0.8%) of postganglionic sympathetic MCG neurons expressed simultaneously both catecholamine-synthesizing enzymes (neurons were TH/DbetaH-positive). A large population of noradrenergic neurons exhibited immunoreactivity (IR) to NPY (62.2 +/- 2.2%), but single NPY-positive perikarya-lacking noradrenergic markers were also observed (2.0 +/- 0.3%). None of the examined MCG neuronal somata contained SP, CGRP, GAL or VIP. A moderate number of noradrenergic nerve fibres located amongst neuronal cell bodies was also found. In small number of these terminals the presence of NPYor GAL (but not CGRP or VIP) was detected. The ovine MCG was numerously innervated with SP-immunoreactive nerve fibres which sometimes formed basket-like formations around postganglionic neurons. The MCG exhibited a sparse CGRP-immunoreactive innervation and lacked VIP-positive nerve terminals. In many aspects the chemical coding of MCG postganglionic neurons and nerve terminals resembles that found in other mammalian cervico-thoracic paravertebral ganglia, but some important species-dependent differences exist. The functional implications of these differences remain to be elucidated.

Origin of the primary efferent neurons projecting to the prostate of the dog.

The retrograde tracing technique of neuronal tracer Fast Blue was used to determine sources of origin of efferent nerve fibers supplying the prostate of the dog. After injection of Fast Blue into the canine prostate retrogradely labelled neurons were found in bilateral L3-S3 sympathetic chain ganglia, bilateral caudal mesenteric ganglion and in bilateral pelvic plexus ganglia. No Fast Blue-positive neurons were present in bilateral L1-L2 sympathetic chain ganglia and in coeliac-mesenteric ganglion complex. The vast majority of Fast Blue-positive efferent prostate-projecting neurons (56.2% +/- 1.7) were located in bilateral caudal mesenteric ganglion, whereas 28.7% +/- 1.5 of them were located in bilateral pelvic plexus ganglia and 14.9% +/- 0.5 in bilateral L3-S3 sympathetic chain ganglia. Immunohistochemical staining for tyrosine hydroxylase and dopamine beta-hydroxylase was applied to determine the neurochemical character of Fast Blue-positive efferent neurons. Immunohistochemistry revealed that in all tyrosine hydroxylase immunoreactive Fast Blue-positive neurons immunoreactivity for dopamine beta-hydroxylase was also found (noradrenergic neurons) while all tyrosine hydroxylase-negative Fast Blue-positive neurons did not express dopamine beta-hydroxylase (non-noradrenergic neurons). In bilateral sympathetic chain ganglia, 96.4% +/- 2.1 of the prostate-projecting neurons were adrenergic and in bilateral caudal mesenteric ganglion this frequency amounted to 95.6% +/- 1.6. In bilateral pelvic plexus ganglia, 26.7% +/- 1.5 of the prostate-supplying efferent neurons did not express either tyrosine hydroxylase or dopamine beta-hydroxylase immunoreactivity which makes discussion of their cholinergic character possible.

Distribution of calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) immunoreactive nerve fibers in the seminal vesicle and prostate of the male sheep.

Double immunohistochemistry was used to determine the occurrence and distribution pattern of nerve fibres immunoreactive to calcitonin gene-related peptide (CGRP), substance P (SP) and galanin (GAL) in seminal vesicles and prostate of the male sheep. Numerous CGRP- and SP-immunoreactive (IR) nerve fibres were found in the mucosal layer and smooth musculature of the seminal vesicles and prostate. In both glands nerve terminals immunoreactive to CGRP were more numerous than SP-IR ones. The majority of CGRP-IR nerve fibers showed colocalization of this peptide and SP. In both layers of the seminal vesicle and prostate, rare nerve terminals immunoreactive to GAL were also found. Immunoreactivity to SP was also found in all GAL-IR nerve fibers. The presence of numerous CGRP- and SP-IR nerve fibers in the seminal vesicle and prostate of the male sheep suggests that these neuropeptides may be involved in the sensory transmission and/or control of smooth muscle contractility. On the other hand, a relatively low number of GAL-IR nerve fibers of the seminal vesicle and prostate suggest that this peptide may act as an anti-nociceptive agent. It cannot be excluded that, in the seminal vesicle, GAL may also be involved in the control of the smooth muscle fiber activity. The possible role of CGRP, SP and GAL in the regulation of functions of the accessory sexual glands needs to be determined in further physiological studies.